GM Crop Database

Database Product Description

676, 678, 680
Host Organism
Zea mays (Maize)
Trait
Glufosinate ammonium herbicide tolerance and fertility restored.
Trait Introduction
Microparticle bombardment of plant cells or tissue
Proposed Use

Production for human consumption and livestock feed.

Product Developer
Pioneer Hi-Bred International Inc.

Summary of Regulatory Approvals

Country Food Feed Environment Notes
United States 1998 1998 1998

Introduction Expand

Maize lines 676, 678, and 680 were developed to provide a reliable hybrid system based on nuclear male sterility. The transgenic lines were produced by genetically engineering plants to be male sterile and tolerant to the herbicide glufosinate ammonium, where herbicide tolerance was used as a selectable marker to identify the male sterile plants. These transformed maize lines contain an introduced gene (dam) from Escherichia coli that encodes the enzyme DNA adenine methylase (DAM). Expression of this gene in specific plant tissues results in the inability of the transformed plants to produce anthers or pollen, resulting in a male sterile plant.

The maize lines 676, 678, and 680 also contained the pat gene, isolated from the bacterium Streptomyces viridochromogenes, for use as a selectable marker. The pat gene encodes a phosphinothricin acetyl transferase (PAT) enzyme, which, when introduced into a plant cell, confers tolerance to the herbicide glufosinate ammonium. The pat gene was used as a selectable marker to identify transformed plants during tissue culture regeneration, and as a field selection method to identify the male sterile lines prior to flowering. Under field conditions, plants that were not male-sterile could be eliminated by application of the herbicide glufosinate ammonium. The novel hybrid system provided an efficient and effective way to identify male-sterile plants for use in hybrid seed production.

Glufosinate ammonium is the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®). Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine. Essentially, glufosinate acts enough like glutamate, the molecule used by glutamine synthetase to make glutamine, that it blocks the enzyme's usual activity. Glutamine synthetase is also involved in ammonia detoxification. The action of glufosinate results in reduced glutamine levels and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell membrane disruption and cessation of photosynthesis resulting in plant withering and death. The PAT enzyme detoxifies phosphinothricin by acetylation into an inactive compound.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
dam DNA adenine methylase MS Maize 512del anther-specific promoter Solanum tuberosum proteinase inhibitor II (pinII) transcription termination signal 1 (676); 3 (678); 1+3 partial (680) Native
pat phosphinothricin N-acetyltransferase HT CaMV 35S 2 (676); 1+1 partial (678); 1 (680) Native

Characteristics of Zea mays (Maize) Expand

Center of Origin Reproduction Toxins Allergenicity

Mesoamerican region, now Mexico and Central America

Cross-pollination via wind-borne pollen is limited, pollen viability is about 30 minutes. Hybridization reported with teosinte species and rarely with members of the genus Tripsacum.

No endogenous toxins or significant levels of antinutritional factors.

Although some reported cases of maize allergy, protein(s) responsible have not been identified.

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Streptomyces viridochromogenes pat

S. viridochromogenes is ubiquitous in the soil. It exhibits very slight antimicrobial activity, is inhibited by streptomycin, and there have been no reports of adverse affects on humans, animals, or plants.

Modification Method Expand

These transgenic maize lines were produced using a microparticle bombardment (biolistic) technique to introduce the genes of interest, followed by plant regeneration in tissue culture on glufosinate-containing medium to select transformed plants. The plasmid DNA, PHP6710, used in the transformation contained three genes: (1) DNA adenine methylase encoding dam gene; (2) the pat gene from S. viridochromogenes; and the beta-lactamase encoding bla gene, which conferred resistance to the antibiotic ampicillin and was used to select bacterial colonies transformed with plasmid DNA.

Tissue-specific expression of the dam gene was accomplished by including sequences from the maize anther-specific 512del promoter, and constitutive expression of the pat gene was regulated using the cauliflower mosaic virus (CaMV) 35S promoter.

Prior to biolistic transformation, PHP6710 plasmid DNA was subjected to restriction enzyme digestion to isolate a 4.5 kb DNA fragment containing the dam and pat genes, but not the bla gene. It was this DNA fragment, not the entire plasmid, that was used for the transformation procedure.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analyses of genomic DNA from each of the male-sterile lines confirmed that all of the lines contained one complete copy of dam and pat at a single insertion site. In addition, each line contained partial and/or rearranged copies of either the pat or dam genes. For example, line 676 contained a second pat gene at a separate insertion site; line 678 contained two additional copies of dam and one additional partial copy of pat; and line 680 contained three additional partial copies of dam. Similar analyses were also used to verify that, as predicted, the antibiotic resistance marker gene (bla) was not incorporated into any of the male-sterile lines.

Genetic Stability of the Introduced Trait

Genetic analysis of maize lines 676, 678, 680 demonstrated that the pat and dam genes segregated as a single genetic locus according to Mendelian rules of inheritance.

Expressed Material

The production of dam-specific mRNA in each of these lines was tested by Northern blot analysis, and detectable amounts were found only in anther tissue (tetrad release stage) obtained from line 678. The inability to detect dam mRNA in similar tissue from the other two lines was attributed to the fact that the expressed DAM enzyme results in cell ablation and destruction.

The levels of PAT protein expression were determined in grain and leaf tissue from each line and found to range between 204-278 µg/g total protein and 601-717 µg/g total protein in leaf tissue from lines 676 and 678, respectively. Concentrations of PAT were below the limit of detection (20 µg/g total protein) in both grain and leaf tissue from line 680. Grain from lines 676 and 678 contained low levels of PAT protein, ranging from undetectable to 19.2 µg/g total protein and 14.8 µg/g total protein, respectively.

Environmental Safety Considerations Expand

Field Testing

The maize lines 676, 678, and 680 have been field tested in the major maize growing regions of the United States since 1995. Field data regarding seed germination rates, yield characteristics, disease and pest susceptibilities, compositional analyses, and numerous other reports supported that conclusion that maize lines 676, 678, and 680 were as safe to grow as any other male sterile maize and would not present a plant pest risk.

Outcrossing

Multiple barriers, including sterility of the maize lines 676, 678, and 680, ensured that gene introgression from these transformed lines into wild or cultivated sexually-compatible plants was extremely unlikely and such rare events would not increase the weediness potential of any resulting progeny or adversely impact biodiversity.

In the United States, where there are few plant species closely-related to maize in the wild, the risk of gene flow to other species is remote. Cultivated maize, Zea mays L. subsp. mays, is sexually compatible with other members of the genus Zea, and to a much lesser degree with members of the genus Tripsacum.

Weediness Potential

It was determined that the relevant introduced trait, male sterility, was unlikely to increase weediness of maize lines 676, 678, and 680. There was no indication that the presence of a specific DNA adenine methylase enzyme, would convert maize into a weed. As well, resistance to glufosinate ammonium would not, in itself, render maize weedy or invasive of natural habitats.

Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. In agriculture, maize volunteers are not uncommon but are easily controlled by mechanical means or by using herbicides, other than glufosinate ammonium. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects

It was determined that genetically modified maize lines 676, 678, and 680 did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and were not expected to impact on threatened or endangered species. Analysis of maize lines 676, 678, and 680 identified no toxic components present in concentrations significantly different from concentrations in nontransgenic maize. Furthermore, the genetic modification in maize lines 676, 678, and 680 did not result in the production of new proteins, enzymes, or metabolites, in the plant, known to have toxic properties, and plants did not exhibit any pathogenic properties.

Impact on Biodiversity

Maize lines 676, 678, and 680 have no novel phenotypic characteristics that would extend their use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in the United States is remote, it was determined that the risk of transferring genetic traits from these maize lines to species in unmanaged environments was insignificant. It was determined that the relative impact on plant biodiversity was neutral, as was the impact on animal and microbe biodiversity since the introduced genes were not expected to alter the plant's metabolism and as such, novel compounds would not be produced.

Food and/or Feed Safety Considerations Expand

Nutritional Data

Samples of grain and forage from transgenic and non-transgenic control lines were subjected to compositional analyses measuring the levels of protein, fat, ash, calcium and phosphorous. Forage samples were also analyzed for crude fibre content. For forage, the levels of analyzed components were found to be comparable between the male-sterile lines, between these lines and non-transgenic control lines, and to previously published literature values. With the exception of calcium levels, which were comparable between the male-sterile and non-transgenic control lines but lower than literature values, similar results were reported for the compositional analyses of grain samples. The lower calcium levels were attributed to an alteration in the analytical method.

Analyses of fatty acids (C16:0, palmitic; C18:0, stearic; C18:1, oleic; C18:2, linoleic; and C18:3, linolenic) and amino acids in samples from each male-sterile line and non-transgenic control lines did not reveal any significant differences in the respective value of each analyte. Except for the levels of a few amino acids, which were lower than those reported in the literature for commercial maize hybrids, the values measured for each analyte were within the range reported in the scientific literature. The differences noted in the levels of a few amino acids were likely due to the genetic background of the inbred parental lines relative to those lines analyzed in previously published studies.

Toxicity and Allergenicity

Previous studies have demonstrated that the PAT protein has a very low potential to be toxic or allergenic. The enzyme possesses none of the physiochemical characteristics commonly associated with known toxins or allergens, such as resistance to thermal or proteolytic degradation. The amino acid sequence of the PAT protein does not share homology with the sequences of known protein toxins and allergens, and in acute toxicity testing with laboratory mammals, adverse effects have never been observed.

Abstract Collapse

Maize (Zea mays L.), or corn, is is grown primarily for its kernel, which is largely refined into products used in a wide range of food, medical, and industrial goods.

Only a small amount of whole maize kernel is consumed by humans. Maize oil is extracted from the germ of the maize kernel and maize is also a raw material in the manufacture of starch. A complex refining process converts the majority of this starch into sweeteners, syrups and fermentation products, including ethanol. Refined maize products, sweeteners, starch, and oil are abundant in processed foods such as breakfast cereals, dairy goods, and chewing gum.
In the United States and Canada maize is typically used as animal feed, with roughly 70% of the crop fed to livestock although an increasing amount is being used for the production ot ethanol. The entire maize plant, the kernels, and several refined products such as glutens and steep liquor, are used in animal feeds. Silage made from the whole maize plant makes up 10-12% of the annual corn acreage, and is a major ruminant feedstuff. Livestock that feed on maize include cattle, pigs, poultry, sheep, goats, fish and companion animals.

Industrial uses for maize products include recycled paper, paints, cosmetics, pharmaceuticals and car parts.

The maize lines 676, 678, and 680 were genetically engineered to express male sterility and tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®). Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine. Essentially, glufosinate acts enough like glutamate, the molecule used by glutamine synthetase to make glutamine, that it blocks the enzyme's usual activity. Glutamine synthetase is also involved in ammonia detoxification. The action of glufosinate results in reduced glutamine levels and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell membrane disruption and cessation of photosynthesis resulting in plant withering and death.

Glufosinate tolerance in these maize lines is the result of introducing a gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT) isolated from the common aerobic soil actinomycete, Streptomyces viridochromogenes, the same organism from which glufosinate was originally isolated. The PAT enzyme catalyzes the acetylation of phosphinothricin, detoxifying it into an inactive compound. The PAT enzyme is not known to have any toxic properties.
The male-sterile trait was introduced by inserting a bacterial gene encoding the enzyme DNA adenine methylase (DAM). Expression of the Escherichia coli dam gene in specific plant tissues results in the inability of the transformed plants to produce anthers or pollen, resulting in a male-sterile plant. The PAT enzyme was used as a selectable marker enabling identification of transformed plants during tissue culture regeneration, and as a field selection method to identify the male-sterile lines prior to flowering. Under field conditions, plants that were not male-sterile could be eliminated by application of the herbicide glufosinate ammonium. The novel hybrid system provided an efficient and effective way to identify male-sterile plants for use in hybrid seed production.

The maize lines 676, 678, and 680 have been field tested in the major maize growing regions of the United States since 1995. Field data regarding seed germination rates, yield characteristics, disease and pest susceptibilities, compositional analyses, and numerous other reports supported the conclusion that maize lines 676, 678, and 680 were as safe to grow as any other male sterile maize and would not present a plant pest risk. It was demonstrated that the transformed maize lines did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. Maize lines 676, 678, and 680 were not expected to impact on threatened or endangered species.

Maize does not have any closely related species growing in the wild in the continental United States and Canada. Cultivated maize can naturally cross with annual teosinte (Zea mays ssp. mexicana) when grown in close proximity, however, these wild maize relatives are native to Central America and are not naturalized in North America. Additionally, multiple barriers, including sterility of the maize lines 676, 678, and 680, ensured that gene flow from these transformed lines into wild or cultivated sexually-compatible plants was extremely unlikely. Gene exchange between maize lines 676, 678, and 680 and maize relatives was determined to be negligible in managed ecosystems, with no potential for transfer to wild species in Canada and the United States.

In an assessment of food and livestock feed safety, forage and grain from these male-sterile lines were analyzed for nutritional composition and compared to the nutritional composition of non-transgenic inbred parental lines. Proximate, fatty acid, and amino acid analyses were performed and in each case there were no significant differences noted between the transgenic and non-transgenic control lines. The use of maize products derived from lines 676, 678, and 680 was not anticipated to have any significant impact on the nutritional quality of the food supply.

Previous studies have demonstrated that the PAT protein has a very low potential to be toxic or allergenic. The enzyme possesses none of the physiochemical characteristics commonly associated with known toxins or allergens, such as resistance to thermal or proteolytic degradation, and its amino acid sequence does not show homology with the sequences of known protein toxins and allergens.

Links to Further Information Expand


This record was last modified on Wednesday, February 25, 2015